M. Eugenia Schininà

Maize polyamine oxidase: primary structure from protein and cDNA sequencing

The first complete amino acid sequence of a flavin‐containing polyamine oxidase was solved by a combined approach of nucleotide and peptide sequence analysis. A cDNA of 1737 bp, isolated from maize seedlings by reverse transcription‐polymerase chain reaction and rapid amplification of cDNA ends strategies, was cloned and its sequence determined. This cDNA contains information for a polypeptide chain of 500 amino acids. Its amino‐terminal sequence shows the typical features of secretion signal peptides. The primary structure of the mature protein was independently confirmed by extensive amino acid sequencing. Structural relationships with flavin‐containing monoamine oxidases are also discussed.

Oxidative stress occurs early in Down syndrome pregnancy: A redox proteomics analysis of amniotic fluid

Purpose: The present study aims to evaluate a set of oxidative stress biomarkers in the amniotic fluid (AF) of women carrying Down syndrome (DS) fetuses that could prove in vivo the early occurrence of oxidative damage in DS.

Involvement of Oxidative Stress in Occurrence of Relapses in Multiple Sclerosis: The Spectrum of Oxidatively Modified Serum Proteins Detected by Proteomics and Redox Proteomics Analysis

Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the central nervous system. Several evidences suggest that MS can be considered a multi-factorial disease in which both genetics and environmental factors are involved. Among proposed candidates, growing results support the involvement of oxidative stress (OS) in MS pathology. The aim of this study was to investigate the role of OS in event of exacerbations in MS on serum of relapsing-remitting (RR-MS) patients, either in relapsing or remitting phase, with respect to serum from healthy subjects. We applied proteomics and redox proteomics approaches to identify differently expressed and oxidatively modified proteins in the low-abundant serum protein fraction. Among differently expressed proteins ceruloplasmin, antithrombin III, clusterin, apolipoprotein E, and complement C3, were up-regulated in MS patients compared with healthy controls. Further by redox proteomics, vitamin D-binding protein showed a progressive trend of oxidation from remission to relapse, respect with controls. Similarly, the increase of oxidation of apolipoprotein A-IV confirmed that levels of OS are elevated with the progression of the disease. Our findings support the involvement of OS in MS and suggest that dysfunction of target proteins occurs upon oxidative damage and correlates with the pathology.

A Strategic Protein in Cytochrome c Maturation THREE-DIMENSIONAL STRUCTURE OF CcmH AND BINDING TO APOCYTOCHROME c

CcmH (cytochromes c maturation protein H) is an essential component of the assembly line necessary for the maturation of c-type cytochromes in the periplasm of Gram-negative bacteria. The protein is a membrane-anchored thiol-oxidoreductase that has been hypothesized to be involved in the recognition and reduction of apocytochrome c, a prerequisite for covalent heme attachment. Here, we present the 1.7Å crystal structure of the soluble periplasmic domain of CcmH from the opportunistic pathogen Pseudomonas aeruginosa (Pa-CcmH*). The protein contains a three-helix bundle, i.e. a fold that is different from that of all other thiol-oxidoreductases reported so far. The catalytic Cys residues of the conserved LRCXXC motif (Cys25 and Cys28), located in a long loop connecting the first two helices, form a disulfide bond in the oxidized enzyme. We have determined the pKa values of these 2 Cys residues of Pa-CcmH* (both >8) and propose a possible mechanistic role for a conserved Ser36 and a water molecule in the active site. The interaction between Pa-CcmH* and Pa-apocyt c551 (where cyt c551 represents cytochrome c551) was characterized in vitro following the binding kinetics by stopped-flow using a Trp-containing fluorescent variant of Pa-CcmH* and a dansylated peptide, mimicking the apocytochrome c551 heme binding motif. The kinetic results show that the protein has a moderate affinity to its apocyt substrate, consistent with the role of Pa-CcmH as an intermediate component of the assembly line for c-type cytochrome biogenesis.

The primary structure of iron superoxide dismutase from Escherichia coli

The complete amino acid sequence of iron superoxide from Escherichia coli has been determined. The sequence was deduced from analysis of peptides obtained after cleavage of the carboxymethylated apoenzyme with trypsin, Stapholococcus aureus protease or CNBr. The polypeptide chain is made up of 192 residues and is easily aligned with the other known amino acid sequences of iron and manganese superoxide dismutases from various sources. The iron superoxide dismutase from E. coli shows a significantly higher homology with the iron enzyme from a different organism than with the manganese isoenzyme from E. coli.

Human Papillomavirus-16 E7 Interacts with Glutathione S-Transferase P1 and Enhances Its Role in Cell Survival

Background Human Papillomavirus (HPV)-16 is a paradigm for “high-risk” HPVs, the causative agents of virtually all cervical carcinomas. HPV E6 and E7 viral genes are usually expressed in these tumors, suggesting key roles for their gene products, the E6 and E7 oncoproteins, in inducing malignant transformation. Methodology/Principal Findings By protein-protein interaction analysis, using mass spectrometry, we identified glutathione S-transferase P1-1 (GSTP1) as a novel cellular partner of the HPV-16 E7 oncoprotein. Following mapping of the region in the HPV-16 E7 sequence that is involved in the interaction, we generated a three-dimensional molecular model of the complex between HPV-16 E7 and GSTP1, and used this to engineer a mutant molecule of HPV-16 E7 with strongly reduced affinity for GSTP1.When expressed in HaCaT human keratinocytes, HPV-16 E7 modified the equilibrium between the oxidized and reduced forms of GSTP1, thereby inhibiting JNK phosphorylation and its ability to induce apoptosis. Using GSTP1-deficient MCF-7 cancer cells and siRNA interference targeting GSTP1 in HaCaT keratinocytes expressing either wild-type or mutant HPV-16 E7, we uncovered a pivotal role for GSTP1 in the pro-survival program elicited by its binding with HPV-16 E7. Conclusions/Significance This study provides further evidence of the transforming abilities of this oncoprotein, setting the groundwork for devising unique molecular tools that can both interfere with the interaction between HPV-16 E7 and GSTP1 and minimize the survival of HPV-16 E7-expressing cancer cells.

HIPK2 Controls Cytokinesis and Prevents Tetraploidization by Phosphorylating Histone H2B at the Midbody

Failure in cytokinesis, the final step in cell division, by generating tetra- and polyploidization promotes chromosomal instability, a hallmark of cancer. Here we show that HIPK2, a kinase involved in cell fate decisions in development and response to stress, controls cytokinesis and prevents tetraploidization through its effects on histone H2B. HIPK2 binds and phosphorylates histone H2B at S14 (H2B-S14(P)), and the two proteins colocalize at the midbody. HIPK2 depletion by targeted gene disruption or RNA interference results in loss of H2B-S14(P) at the midbody, prevention of cell cleavage, and tetra- and polyploidization. In HIPK2 null cells, restoration of wild-type HIPK2 activity or expression of a phosphomimetic H2B-S14D derivative abolishes cytokinesis defects and rescues cell proliferation, showing that H2B-S14(P) is required for a faithful cytokinesis. Overall, our data uncover mechanisms of a critical HIPK2 function in cytokinesis and in the prevention of tetraploidization.

Cystamine restores GSTA3 levels in Vanin-1 null mice.

Free cysteamine levels in mouse tissues have been strictly correlated to the presence of membrane-bound pantetheinase activity encoded by Vanin-1. Vanin-1 is involved in many biological processes in mouse, from thymus homing to sexual development. Vanin-1 -/- mice are fertile and grow and develop normally; they better control inflammation and most of the knockout effects were rescued by cystamine treatment. Gene structure analysis showed the presence of an oxidative stimuli-responsive ARE-like sequence in the promoter. In this paper we investigate antioxidant-detoxifying enzymatic activities at the tissue level, comparing Vanin-1 -/- and wild-type mice. In Vanin-1 null animals we pointed out a decrease in the Se-independent glutathione peroxidase activity. The decrease in enzymatic activity appeared to be correlated to an impairment of GST isoenzyme levels. In particular a significant drop in GSTA3 together with a minor decrement in GSTM1 and an increase in GSTP1 levels was detected in Vanin-1 -/- livers. Cystamine administration to Vanin-1 -/- mice restored specifically GSTA3 levels and the corresponding enzymatic activity without influencing protein expression. A possible role of cystamine on protein stability/folding can be postulated.

Anti-Proliferative Effect of Rosmarinus officinalis L. Extract on Human Melanoma A375 Cells

Rosemary (Rosmarinus officinalis L.) has been used since ancient times in traditional medicine, while nowadays various rosemary formulations are increasingly exploited by alternative medicine to cure or prevent a wide range of health disorders. Rosemary’s bioproperties have prompted scientific investigation, which allowed us to ascertain antioxidant, anti-inflammatory, cytostatic, and cytotoxic activities of crude extracts or of pure components. Although there is a growing body of experimental work, information about rosemary’s anticancer properties, such as chemoprotective or anti-proliferative effects on cancer cells, is very poor, especially concerning the mechanism of action. Melanoma is a skin tumor whose diffusion is rapidly increasing in the world and whose malignancy is reinforced by its high resistance to cytotoxic agents; hence the availability of new cytotoxic drugs would be very helpful to improve melanoma prognosis. Here we report on the effect of a rosemary hydroalcoholic extract on the viability of the human melanoma A375 cell line. Main components of rosemary extract were identified by liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) and the effect of the crude extract or of pure components on the proliferation of cancer cells was tested by MTT and Trypan blue assays. The effect on cell cycle was investigated by using flow cytometry, and the alteration of the cellular redox state was evaluated by intracellular ROS levels and protein carbonylation analysis. Furthermore, in order to get information about the molecular mechanisms of cytotoxicity, a comparative proteomic investigation was performed.

Proteomics analysis of protein expression and specific protein oxidation in human papillomavirus transformed keratinocytes upon UVB irradiation

Increasing evidence supports the role of oxidative stress in cancer development. Ultraviolet (UV) irradiation is one of the major sources of oxidative stress through the generation of reactive oxygen species (ROS). Besides the physiological function of ROS in cellular homeostasis, accumulating reports suggest that ROS are involved in all stages of multistep cancer development. In order to investigate the involvement of oxidative damage into the mechanisms of tumour progression, we used a parallel proteomic approach to analyse the protein expression profile and to identify oxidatively modified proteins in human papillomavirus (HPV)‐transformed keratinocytes (HK‐168 cells) upon ultraviolet B (UVB) exposure. The HK‐168 cells were obtained from normal human epidermal keratinocytes transfected with the whole genome of the high‐risk HPV type 16, unanimously recognized as an etiological agent of cervical carcinoma. Because of its year‐long latency, this tumour offers a convenient model to study the role of environmental concurring agents in the multistep malignant progression. By the protein expression profile, we identified 21 proteins that showed different expression levels in HK‐168 cells treated with UVB in comparison with untreated cells. Focusing on the oxidative modifications occurring at the protein level, we identified five proteins that showed elevated protein carbonyls levels: α‐enolase, heat shock protein 75, annexin 2, elongation factor Tu and elongation factor γ. Our results indicate that UVB‐induced oxidative stress perturbs the normal redox balance and shifts HPV‐transformed keratinocytes into a state in which the carbonylation of specific proteins is systematically induced. We suggest that UVB‐induced modulation of protein expression combined with oxidative modification lead to protein dysfunction that might contribute to the malignant progression of transformed cells.